Single-Cell RNA Sequencing of Circulating Myeloma Cells: Clinical Implications in Times of Immunotherapies

Introduction

In the times of promising immunotherapies against multiple myeloma (MM), close monitoring of patients’ response and disease complexity is crucial for accurate decision making. To date, bone marrow (BM) aspiration and biopsy have been the gold standard for diagnosis and molecular characterization of the disease. These procedures, however, are invasive and associated with discomfort for the patients. Not all existing disease clones can be captured by a one-time aspiration at a single location. Furthermore, current standard diagnostic methods such as FiSH and bulk DNA sequencing provide little information with regard to decision making about treatment with modern therapeutics such as monoclonal antibodies, bispecific T-cell engagers, and CAR-T cells. Here, we present data to show that single-cell RNA sequencing (scRNA-seq) of circulating MM cells is an excellent proxy for detecting prognostically relevant inferred copy number variants (CNVs) and translocations, while depicting the heterogeneity of the disease. Moreover, scRNA-seq can yield additional, therapeutically relevant information such as expression level of the target protein for the immunotherapies. Because of the non-invasiveness and possibility of frequent peripheral blood (PB) draws, this method constitutes a great tool for patient monitoring in both clinical-trial and non-trial settings.

Methods

Fresh PB and BM samples of 10 myeloma patients at various disease stages were obtained. Mononuclear cells were isolated by density-gradient centrifugation. CD138-positive cells were selected using magnetic-beads separation. Single MM cells were sorted onto 96-well plates using the following gating strategy: 7AAD-, CD14-, CD138+, CD38+, SLAMF7+, CD45-/CD45^dim. RNA isolation and cDNA-library preparation were performed using the Smart-seq2 method (Picelli et al. Nat Prot 2014), which yielded information of the full transcript length.

For testing purposes, we used our previously published data (Frede et al. Nat Cell Biol 2021). Clustering was performed with Seurat. Cell-type annotation was performed using SingleR and reference dataset from the BLUEPRINT consortium. Light- and heavy-chain B-cell receptor sequences were determined using BASIC and igBLAST. CNV analysis was performed with inferCNV. Expression level was assessed for genes expected to be overexpressed in case of translocations (CCND1, CCND3, MAF, MAFB, MMSET, FGFR3) as well as for surface marker genes (CD138, CD38, SLAMF7, BCMA, GPRC5D, FCRH5). Fisher test (expression of a particular gene vs. all other genes without the gene of interest) was performed to define the p-value cut-off for identifying an inferred translocation. Percentage of MM cells expressing a surface marker was assessed for each sample.

Results

We could reproduce the heavy- and light-chain clonality with a sensitivity and specificity of 100 %. scRNA-seq showed to be a valid substitute for BM FiSH with a sensitivity of 94.4 % and specificity of 100 % in detecting prognostically relevant CNVs (amp/gain(1q), del(1p), del(17p), hyperdiploidy) and translocations (t(11;14), t(6;14), t(14;16), t(14;20), t(4;14)).

Interestingly, we could show a direct correlation between very low expression of BCMA (expression in 8.1% of circulating MM cells) 29 months after 1^st CAR-T cell therapy and progressive disease directly after re-exposure to a 2^nd dose of anti-BCMA CAR-T cells (31 months after the 1^st administration), suggesting that circulating tumor cells may aid in identifying effective therapies and avoiding those that are not. Furthermore, we observed 2 patients with significant downregulation of BCMA 1.5 months and 14 months (10.5 % and 3 %, respectively) after disease progression under belantamab mafodotin as well as a primary low expression (8.6 %) of FcRH5 in a patient naïve to anti-FcRH5 treatment. Moreover, in 2 patients, FiSH from BM was not feasible due to technical difficulties, which further highlights the utility of interrogation of single MM cells from PB.

Conclusion

scRNA-seq of MM cells from PB is a robust, non-invasive substitute for BM FiSH to detect prognostically and therapeutically relevant CNVs and translocations. Furthermore, it yields additional information such as expression level or absence of surface markers as immunotherapeutic targets. This is of critical use in clinical decision making and provides opportunity for more personalized therapy approaches.